HSP70-9 Antibody

What is HSP70-9 and why is it important in cellular function?

HSP70-9 (HSPA9/mortalin) is a mitochondrial chaperone protein belonging to the heat shock protein 70 family. It plays crucial roles in various cellular processes including protein folding, stress response, mitochondrial import, and cellular homeostasis. The protein is highly conserved across species, indicating its fundamental importance in cellular function. HSP70-9 has been implicated in diverse physiological processes, which explains why it has been known under several different names in scientific literature, including heat-shock 70 kDa protein 9 . Dysregulation of HSP70 family proteins has been linked to various diseases, including cancer, neurodegenerative disorders, and cardiovascular conditions, making them important research targets for potential therapeutic interventions .

What are the key applications for HSP70-9 antibodies in research?

HSP70-9 antibodies serve multiple research applications:

• Western blotting: For detecting and quantifying HSP70-9 expression levels in various cell types and tissues

• Immunohistochemistry/Immunofluorescence: For localizing HSP70-9 within cells and tissues

• Immunoprecipitation: For studying protein-protein interactions

• ELISA: For quantitative measurement of HSP70-9 in samples

• Flow cytometry: For analyzing HSP70-9 expression at the single-cell level

Most HSP70 antibodies are validated for applications including Western blot (WB), immunofluorescence/immunocytochemistry (IF/ICC), and ELISA as demonstrated with products like CAB4777 . When selecting an antibody, researchers should confirm its validation for their specific application and target species.

How can I optimize Western blot protocols when using HSP70-9 antibodies?

For optimal Western blot results with HSP70-9 antibodies:

• Sample preparation: Include protease inhibitors to prevent degradation

• Loading control: Use appropriate controls like β-actin or GAPDH

• Antibody dilution: Follow manufacturer recommendations, typically starting at 1:500-1:2000 for primary antibodies like CAB4777

• Incubation conditions: Optimize time and temperature (typically overnight at 4°C)

• Detection system: Choose appropriate secondary antibodies and detection method

• Validation: Verify antibody specificity using positive control samples such as HeLa, HepG2, or Jurkat cell lines

Certificate of Analysis data shows that 1 μg/ml of some HSP70 antibodies is sufficient for detection in 20 μg of heat-shocked HeLa cell lysate using colorimetric immunoblot analysis .

What are the recommended storage conditions for HSP70-9 antibodies?

Most HSP70 antibodies require specific storage conditions to maintain activity:

Always check the certificate of analysis and product documentation for specific storage requirements . Improper storage can significantly impact antibody performance in experimental applications.

How can I distinguish between different HSP70 family members when using antibodies?

Distinguishing between highly homologous HSP70 family members requires careful antibody selection and experimental design:

• Epitope specificity: Select antibodies raised against unique epitopes - for example, some HSP70-9 antibodies target specific sequences not present in other family members

• Cross-reactivity testing: Validate using knockout/knockdown samples or recombinant proteins

• Molecular weight verification: HSP70-9 (~74 kDa) can sometimes be distinguished from other family members by precise molecular weight

• Subcellular fractionation: HSP70-9 predominantly localizes to mitochondria, unlike some family members

• Sequential immunoprecipitation: To separate specific family members

When working with multiple HSP70 family proteins, carefully review antibody documentation for specificity information. Some antibodies like BB70 detect both HSP70 (~72 kDa) and HSC70 (~73 kDa) , which could complicate interpretation when studying HSP70-9 specifically.

What experimental controls should be included when studying extracellular HSP70-9?

When investigating extracellular HSP70-9, include these essential controls:

• Endotoxin contamination control: HSP70 studies have been complicated by potential endotoxin contamination that can confound results

• Immunodepletion controls: As demonstrated in studies where culture supernatants were incubated with anti-human HSP70 monoclonal antibody (1:100 dilution) or control mouse IgG, followed by protein A-agarose incubation to confirm specificity of observed effects

• Cell viability assessment: To ensure detected extracellular HSP70-9 isn't from cell lysis

• Time-course sampling: To distinguish active secretion from passive release

• Subcellular fraction markers: To verify purity of extracellular preparations

• Recombinant protein controls: For calibration and specificity validation

Research has shown that extracellular HSP70 can influence processes like MMP-9 expression, but proper controls are essential to confirm these effects are specific to HSP70 and not contaminants .

How do I resolve contradictory results when targeting HSP70-9 in autoimmune disease research?

Research suggests HSP70 can exhibit dual roles in autoimmune diseases, which may explain contradictory results . To address these contradictions:

• Context-specific analysis: Evaluate the specific disease model, tissue location, and experimental conditions

• Intra- vs. extracellular effects: Separately analyze the roles of intracellular and extracellular HSP70-9

• Detailed immunophenotyping: As demonstrated in studies showing anti-HSP70 treatment affecting Th17 cells without significant changes in FoxP3+ or CD4+CD25+ cell frequencies

• Temporal considerations: Examine timing of HSP70-9 targeting relative to disease progression

• Dosage response curves: Test multiple concentrations of antibodies or inhibitors

• Genetic approaches: Complement antibody studies with knockout/knockdown experiments

Studies have shown that anti-HSP70 antibody treatment can decrease pro-inflammatory T cells, specifically reducing splenic Th17 cells while increasing the CD4+FoxP3+:Th17 ratio, providing a potential explanation for therapeutic effects in conditions like psoriasis .

What methodological approaches can improve HSP70-9 antibody validation for research?

Comprehensive validation of HSP70-9 antibodies should include:

• Multiple application testing: Validate across different methods (WB, IF, IP, ELISA)

• Knockout/knockdown controls: Use CRISPR or siRNA approaches to create negative controls

• Epitope mapping: Confirm the specific region recognized by the antibody

• Cross-species reactivity verification: Test in multiple species when claimed (e.g., human, mouse, rat)

• Batch-to-batch consistency testing: Compare performance across different lots

• Independent antibody comparison: Test multiple antibodies targeting different epitopes

• Mass spectrometry validation: For definitive target identification

For example, the CAB4777 antibody is validated as reactive with human, mouse, and rat samples and targets a specific sequence corresponding to amino acids 441-641 of human HSP70 , providing researchers confidence in its specificity.

How can ELISA be optimized for detecting HSP70-9 in clinical samples?

For optimizing HSP70-9 ELISA in clinical research:

• Coating optimization: Use purified HSP70-9 protein at appropriate concentration (e.g., 0.5 μg/ml in 0.1 M bicarbonate buffer) for 18 hours at 4°C

• Blocking protocol: Implement effective blocking (e.g., 1% bovine serum albumin in PBS) for 2 hours at room temperature

• Sample dilution series: Determine optimal dilution for your sample type

• Standard curve development: Create a standard curve using recombinant HSP70-9

• Cross-reactivity testing: Verify specificity against other HSP70 family members

• Batch processing: Process all comparable samples in the same batch

• Spike-in recovery: Add known quantities of recombinant protein to verify detection

Follow protocols similar to published methods, such as using HRP-conjugated secondary antibodies (1:5000 dilution) and TMB substrate solution for visualization . These methodological details are crucial for reproducible quantification of HSP70-9 in clinical samples.

How can HSP70-9 antibodies be utilized in cancer research?

HSP70-9 antibodies provide valuable tools for cancer research applications:

• Expression profiling: HSP70 family proteins are frequently dysregulated in various cancers

• Biomarker development: Evaluate HSP70-9 as a potential diagnostic or prognostic marker

• Therapeutic target validation: Investigate the effects of HSP70-9 inhibition on cancer cell survival and proliferation

• Mechanism studies: Explore HSP70-9's role in cancer-related processes including apoptosis resistance, metastasis, and treatment response

• Drug development: Screen compounds that modulate HSP70-9 function

Research using tools like anti-HSP70 antibodies has revealed connections between HSP70 expression and cancer progression, making this a promising area for continued investigation .

What are the methodological considerations for using HSP70-9 antibodies in neurodegenerative disease research?

When applying HSP70-9 antibodies to neurodegenerative disease research:

• Tissue-specific optimization: Brain tissue often requires specialized fixation and processing protocols

• Cross-blood-brain barrier considerations: For in vivo applications

• Age-matched controls: Essential for age-related neurodegenerative conditions

• Co-localization studies: With disease-specific markers (e.g., amyloid-β, tau, α-synuclein)

• Stress response differentiation: Distinguish between general stress responses and disease-specific effects

• Post-mortem stability assessment: Validate antibody performance in post-mortem tissues

The universal ability of HSP70s to undergo cycles of binding to and release from hydrophobic stretches of partially unfolded proteins makes them particularly relevant to neurodegenerative diseases characterized by protein misfolding and aggregation.

How can contradictory findings about HSP70-9's role in autoimmune diseases be reconciled methodologically?

To reconcile contradictory findings in autoimmune disease studies:

• Dual role analysis: Systematically evaluate both pro- and anti-inflammatory effects under different conditions

• Cell type-specific effects: Analyze HSP70-9 function in different immune cell populations

• Disease stage consideration: Examine effects at different stages of disease progression

• Route of administration impact: Consider how different delivery methods affect outcomes

• Antibody isotype effects: Compare different antibody isotypes when targeting HSP70-9

• Genetic background influence: Test in multiple strain backgrounds

Research has demonstrated that anti-HSP70 treatment can decrease the percentage of pro-inflammatory Th17 cells while increasing the CD4+FoxP3+:Th17 ratio, suggesting immunomodulatory effects that may explain some therapeutic benefits in conditions like psoriasis .

How can non-specific binding be minimized when using HSP70-9 antibodies?

To reduce non-specific binding in HSP70-9 antibody applications:

• Optimize blocking: Use appropriate blocking agents (BSA, non-fat milk, or commercial blockers)

• Antibody titration: Determine optimal concentration through dilution series (e.g., 1:500-1:2000 for Western blot applications)

• Increase washing stringency: Use appropriate detergents and sufficient washing steps

• Pre-adsorption: Consider pre-adsorbing antibodies with related proteins

• Alternative secondary antibodies: Test different secondary antibodies or detection systems

• Sample preparation optimization: Ensure complete denaturation for Western blotting

• Negative controls: Include isotype controls matching the primary antibody

Proper optimization reduces background and increases signal-to-noise ratio, improving the reliability of results when studying HSP70-9.

What strategies help resolve contradictory Western blot results with HSP70-9 antibodies?

When facing contradictory Western blot results:

• Multiple antibody verification: Use antibodies targeting different epitopes of HSP70-9

• Positive control inclusion: Use samples with confirmed HSP70-9 expression (e.g., HeLa, HepG2, Jurkat cells)

• Loading control normalization: Ensure consistent loading with housekeeping proteins

• Denaturation condition testing: Try different sample preparation methods

• Transfer efficiency verification: Use staining methods to confirm protein transfer

• Detection system comparison: Compare chemiluminescence, fluorescence, and colorimetric methods

• Membrane type evaluation: Test PVDF versus nitrocellulose membranes

Certificate of Analysis data showing that 1 μg/ml of HSP70 antibody is sufficient for detection in heat-shocked HeLa cell lysate can serve as a reference point for optimization .

How might HSP70-9 antibodies contribute to developing novel therapeutic approaches?

HSP70-9 antibodies could advance therapeutic development through:

• Target validation: Confirming HSP70-9's role in disease pathogenesis

• Mechanism elucidation: Clarifying how HSP70-9 influences disease processes

• Biomarker identification: Developing diagnostic or prognostic tools

• Patient stratification: Identifying which patients might benefit from HSP70-targeted therapies

• Therapeutic antibody development: Potentially developing antibodies as direct therapeutics

Studies suggest HSP70 may be a promising therapeutic target in psoriasis and potentially other autoimmune dermatoses , illustrating the translational potential of HSP70 family research.

What emerging technologies might enhance HSP70-9 antibody applications in research?

Emerging technologies poised to advance HSP70-9 antibody applications include:

• Single-cell proteomics: For cellular heterogeneity analysis

• Proximity labeling: To identify HSP70-9 interaction partners

• Super-resolution microscopy: For detailed subcellular localization studies

• In vivo imaging: Using labeled antibodies for real-time tracking

• Antibody engineering: Developing more specific variants with enhanced properties

• CRISPR screens: For systematic functional studies complementing antibody approaches

• Artificial intelligence analysis: For pattern recognition in complex datasets

These technologies will enable researchers to address more sophisticated questions about HSP70-9's roles in normal physiology and disease states.